A small heat shock protein stably binds heat-denatured model substrates and can maintain a substrate in a folding-competent state

Abstract

The small heat shock proteins (sHSPs) recently have been reported to have molecular chaperone activity in vitro; however, the mechanism of this activity is poorly defined. We found that HSP18.1, a dodecameric sHSP from pea, prevented the aggregation of malate dehydrogenase (MDH) and glyceraldehyde-3-phosphate dehydrogenase heated to 45 degrees C. Under conditions in which HSP18.1 prevented aggregation of substrates, size-exclusion chromatography and electron microscopy revealed that denatured substrates coated the HSP18.1 dodecamers to form expanded complexes. SDS-PAGE of isolated complexes demonstrated that each HSP18.1 dodecamer can bind the equivalent of 12 MDH monomers, indicating that HSP18.1 has a large capacity for non-native substrates compared with other known molecular chaperones. Photoincorporation of the hydrophobic probe 1,1'-bi(4-anilino)naphthalene-5,5'-disulfonic acid (bis-ANS) into a conserved C-terminal region of HSP18.1 increased reversibly with increasing temperature, but was blocked by prior binding of MDH, suggesting that bis-ANS incorporates proximal to substrate binding regions and that substrate-HSP18.1 interactions are hydrophobic. We also show that heat-denatured firefly luciferase bound to HSP18.1, in contrast to heat-aggregated luciferase, can be reactivated in the presence of rabbit reticulocyte or wheat germ extracts in an ATP-dependent process. These data support a model in which sHSPs prevent protein aggregation and facilitate substrate refolding in conjunction with other molecular chaperones.

Specific protein homeostatic functions of small heat‐shock proteins increase lifespan

“…In vitro , the addition of the HSP70/HSP40 refolding machinery is required for the refolding reaction (Lee et al ., 1997). This has been reproduced in living cells using luciferase as a substrate (Nollen et al ., 2001; Bryantsev et al ., 2007).…”

Differential regulation of HSP27 oligomerization in tumor cells grown in vitro and in vivo

“…the observation that the size of HSP27 oligomers transiently increases in HeLa and L929 cells treated with TNF-a which induces rapid phosphorylation of the protein (Mehlen et al, 1995b(Mehlen et al, ,c, 1997. However, these observations were di cult to interprete as di erent populations of sHSP could exist in these cell lines and the methods used to detect larger oligomers could also identify small oligomers associated with unfolded proteins (Ehrnsperger et al, 1997;Lee et al, 1997). Our conclusion is based on the observation that the aspartate mutant of HSP27 forms large oligomers in REG cells cultured at con¯uence or forming tumors when injected subcutaneously in rodents.…”

Accumulation of small heat shock proteins, including mitochondrial HSP22, induced by oxidative stress and adaptive response in tomato cells

“…Eukaryotic smHSP are considered to be molecular chaperones (Arrigo and Landry, 1994). Chaperone activity for plant smHSP was demonstrated in vitro (for example Lee et al, 1997) and suggested in vivo (reviewed by Waters et al, 1996). Great differences exist, however, between mammalian and plant smHSP with respect to the number of expressed proteins and their localization, and their ability to be regulated by phosphorylation.…”